CN114430810A - Stacked imaging system and method for generating images - Google Patents
Stacked imaging system and method for generating images Download PDFInfo
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- CN114430810A CN114430810A CN201980100600.6A CN201980100600A CN114430810A CN 114430810 A CN114430810 A CN 114430810A CN 201980100600 A CN201980100600 A CN 201980100600A CN 114430810 A CN114430810 A CN 114430810A
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- G02B21/00—Microscopes
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- G02B21/365—Control or image processing arrangements for digital or video microscopes
- G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
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- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
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- G—PHYSICS
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- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
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Abstract
The present invention relates to a stacked imaging system comprising a plurality of light sources adapted to emit light onto a sample location, wherein the light sources are arranged in a predetermined pattern; a controller adapted to control operation of the plurality of light sources; wherein at least one of a) the predetermined pattern of light sources and b) the control operation of the plurality of light sources is adapted to compensate for geometrical effects due to the arrangement of the light sources with respect to the sample position.
Description
Technical Field
The present invention relates to a stacked imaging system and a method of generating an image using a stacked imaging system. In particular, the present invention relates to a fourier stack imaging system and a method of generating an image using a fourier stack imaging system.
Background
An optical system with a high Numerical Aperture (NA) is required for imaging small structures. Digital imaging and fast computer systems provide the opportunity to generate high resolution images by analytically combining low resolution sub-images.
Computational methods of these microscopic imaging include overlay imaging, which generates images by processing coherent interference patterns scattered from a sample. The sample or wave field moves relative to a constant function such as an illumination field or an aperture stop. Fourier stack imaging systems are advantageous for having a large field of view and high resolution. A large number of individual images are illuminated by different numerical aperture settings. Quasi-collimated and hence (spatially) coherent illumination is used at large angles of incidence. From the perspective of the imaging lens, this corresponds to an illumination system with a single asymmetric subset at high numerical aperture settings.
Conventional fourier stacked imaging systems use planar two-dimensional arrays of Light Emitting Devices (LEDs). The light sources are arranged at the same pitch in the x and y directions, i.e. the distance between adjacent LEDs in the x and y directions is kept constant. One light source at a time is turned on and a corresponding low resolution image is captured. The position of the light source relative to the sample determines the measured portion of the fourier spectrum. For fourier stack imaging reconstruction, it is required to collect fourier spectra with at least 50% overlap.
Conventional uniform LED arrays have the effect that due to the high angle of incidence, a geometric reduction of the available power density at the sample plane becomes relevant. The reduction is proportional to the cosine of the angle of incidence and has a significant effect at high angles of incidence.
Another effect is to provide redundant information away from the light source passing through the central optical axis of the sample. As a possible solution, non-uniform sampling is proposed in Guo et al, "Optimization of sampling pattern and the design of Fourier ptychographic illuminator" (sample pattern Optimization and Fourier stacked imaging illuminator design), Opt.Express 23,6171-6180, 2015.
Another approach involves a regular arrangement in polar coordinates as proposed in US 2017/0371141 a 1.
Furthermore, it is known to increase the amount of processing, i.e. to reduce the number of images required for reconstruction, by capturing low resolution images using multiple light sources simultaneously. For example, US 2017/0146788 a1 relates to fourier stack imaging microscopy with multiplexed illumination. US 2016/0088205 a1 relates to a multiplexed fourier stack imaging system and method. Another approach is known from "Computational amplification for high-speed in vitro Fourier ptychographic microscopy" by Tian et al, Optica 2(10), 904-911, 2015.
Another method of increasing the amount of processing is Content adaptive illumination, as known from "Content adaptive illumination for Fourier ptychographic" of Bian et al, opt. lett.39, 6648-6651, 2014. According to the method, the relevant fourier components are identified and the corresponding light source is selected for scanning. By using only a subset of the images, the number of images required can be reduced to some extent.
Because fourier stack imaging systems require the generation of multiple images at successive points in time, there is always a need to further reduce the time required to generate all sub-images that are combined using a stitching algorithm. At the same time, the image quality must be kept high, i.e. the reduction in the time for generating the sub-images should not lead to a significant deterioration of the image quality.
It is therefore an object of the present invention to provide a stack imaging system and a method of generating images using a stack imaging system that generates high quality images in a relatively short time.
Disclosure of Invention
This object is solved by the subject matter of the independent claims. The present invention provides a stack imaging system as recited in claim 1 and a method of generating an image using a stack imaging system as recited in claim 15. Advantageous embodiments are set forth in the dependent claims.
Thus, according to a first aspect, the present invention provides a stacked imaging system having a plurality of light sources and a controller. The light source emits light onto the sample location. The light sources are arranged in a predetermined pattern. The controller controls the operation of the plurality of light sources. The predetermined pattern of light sources is chosen such that geometrical effects due to the arrangement of the light sources with respect to the sample position, which would otherwise occur in a plane-uniform arrangement, are at least partially compensated. Additionally or alternatively, the control operation of the light source is adapted to compensate for geometrical effects due to the arrangement of the light source relative to the sample position.
According to a second aspect, there is provided a method for generating an image using a stacked imaging system, comprising a plurality of light sources arranged in a predetermined pattern. A plurality of light sources emit light onto a sample location including a sample. The controller controls the operation of the plurality of light sources. The predetermined pattern of light sources and/or the control operation of the plurality of light sources is adapted to compensate for geometrical effects due to the arrangement of the light sources with respect to the sample position.
The optimized arrangement of the light sources may increase the throughput of the stacked imaging system compared to conventional fourier stacked imaging systems with consistent spacing and sequential acquisition. Compensation for geometric effects may include compensation for distance effects and angle effects, and may result in more constant and improved signal-to-noise performance over the entire angular spectrum. The overall noise characteristics in the reconstructed image will be improved. The preferred settings may be a constant noise and background level for all sub-images combined in the respective reconstructions.
It is an advantage of the invention that adaptive illumination and/or detection settings, such as the arrangement of light sources and/or the brightness of light sources, provide an optimized signal-to-noise ratio in the sub-images. Furthermore, due to the optimized illumination and exposure control, an optimized image contrast in the sub-images can be achieved. Stacked imaging systems offer greater variability and flexibility. In addition, different diffraction angles may be selectively weighted and compared. According to an embodiment, certain structures, in particular small structures and/or edges and/or transition regions may be enhanced, for example by providing a higher contrast. This effect is particularly advantageous for taking pictures that are further processed by some systems, for example using machine learning methods based on artificial intelligence or neural networks. These systems work better if the contrast is enhanced. In these cases, the natural appearance is less or not important.
Preferably, a larger diffraction angle will be enhanced with respect to the central optical axis. The larger contrast images may be particularly advantageous for applications such as laboratory diagnostics, for example, for hematology of samples that can be imaged multiple times, such as fixed cells on a slide. The present invention provides high resolution imaging with high working distance and large depth of field for reconstructed images typically used in stacked imaging systems.
As used in the present invention, the geometric effect to be compensated may comprise at least one of:
a) angular dependent spatial emission characteristics of the light source, such as angular emission characteristics based on a profile of the geometric arrangement of the light source relative to the sample position.
b) The cosine effect, the proportional relationship of the available power density at the sample location to the cosine of the angle of incidence. The angle of incidence or azimuth of the light source is measured between the central optical axis and the line from the light source to the sample location, i.e., the optical axis of the particular light source. For a planar arrangement of light sources, the central optical axis of the light source arrangement is perpendicular to the plane and passes through the sample position. The arrangement of the light sources may be symmetrical with respect to the central optical axis, for example in the form of a spherical cap. In this case, the arrangement of the light sources selects the central optical axis.
c) The drop in power level caused by the light source illuminating the target area under investigation is kept constant at least at points on the optical axis by suitable control of the light source and/or by its geometrical arrangement close to the spherical arrangement of the light source.
d) Higher diffraction angles result in lower diffraction efficiencies. Compensation for lower diffraction efficiency may result in better resolution and/or higher contrast of small object features in the corresponding reconstructed images. In contrast to high numerical aperture microscopy, stack imaging offers the opportunity to control this effect more effectively than illumination systems with planar illumination profiles used in standard microscopy with kohler-type illumination.
As used in the present invention, a sub-image refers to an image generated using a particular light source or using a particular selection or subset of light sources of a plurality of light sources.
According to another embodiment of the stacked imaging system, the predetermined pattern of the plurality of light sources comprises the light sources arranged in a plurality of concentric rings around a central optical axis passing through the sample location. The variation in azimuth angle is substantially uniform for the light sources in subsequent rings. The change in polar angle is substantially uniform for adjacent light sources within the same concentric ring. According to this embodiment, a stacked imaging system includes a uniform theta-phi illuminator, theta being the polar angle and phi being the azimuthal angle. This particular arrangement of light sources eliminates any variable redundancy of measurements compared to conventional lighting methods. Each subsequent light source provides a consistent angular spacing variation in the radial and azimuthal directions. Preferably, the variation in azimuth and the variation in polar angle are chosen to provide a consistent 50% overlap for all light sources throughout fourier space. The described arrangement reduces the number of images required for reconstruction, resulting in higher throughput. The reconstructed image is free of artifacts generated due to the regular grid arrangement of light sources.
According to another embodiment of the stacked imaging system, the arrangement of the light sources is planar.
According to another embodiment of the stacked imaging system, the arrangement of light sources is spherical. According to this arrangement, the distance between the light source and the sample position is preferably constant. This arrangement has the additional advantage of providing a high dynamic range image for the edge-located light sources. This results in a better signal-to-noise ratio of the reconstructed image. This arrangement can correct for geometric effects caused by directionality and distance between the light source and the sample.
According to another embodiment of the stacked imaging system, the controller controls operation of the plurality of light sources by simultaneously operating the plurality of light sources. The number of light sources to be operated simultaneously is limited by a given maximum number and/or minimum distance criterion in the spatial or angular coordinate space of the light sources. For example, if a stacked imaging system includes multiple N light sources, the number of images required is reduced from N to N/M if multiple M light sources are used simultaneously. This results in an M-fold increase in throughput.
Multiplexing, i.e. operating a plurality of light sources to reduce the number of frames required, may be advantageous as long as no relevant information is lost, i.e. if the different diffraction patterns in the fourier plane can still be distinguished. That is, it should be possible to identify structural regions in fourier space and to limit these regions relative to each other in order to assign them to the respective light sources. Thus, a plurality of diffraction patterns can be separated and extracted from a single frame. To ensure separability, the signals or structures in fourier space must decay fast enough so that the different signals or structures do not combine above the noise level. This can be achieved by a minimum angular distance, i.e. by a minimum distance criterion in the coordinate space of the angle. The minimum distance criterion may be selected based on the structural dimensions of the object to be analyzed. Multiplexing is particularly advantageous for weak diffractive structures with rapidly decaying amplitude distributions in fourier space. Preferably, this distance is maximized, taking into account that diametrically opposed light sources may have overlapping diffractions.
According to another embodiment of the stacked imaging system, the controller controls operation of the plurality of light sources by selecting a subset of the light sources of the plurality of light sources and operating only the light sources of the subset. In fourier slice imaging, instead of scanning the sample in the spatial domain, scanning is performed in the fourier domain. Thus, it is determined which fourier components are present and the corresponding light device is identified. Only the identified light device is used to scan the sample. Because all sub-images are generated in a sequential order, using only a subset of the light sources significantly reduces the time required to generate a high resolution image.
According to another embodiment of the stacked imaging system, the subset of light sources to be operated on is selected based on characteristics of the sample to be observed. It will be appreciated that the sample contains only certain elements or structures. Which fourier component dominates depends on the size and material properties of the element or structure. Thus, only the relevant fourier components, i.e. only the relevant light sources, may be selected based on the characteristics of the sample. For example, if the sample is a blood sample, the controller may select the light source that is most relevant to the blood sample.
According to another embodiment of the stacked imaging system, the subset of light sources to be operated is selected based on a user input. The user may select between different types of samples. The light source for each sample may be stored in the memory of the controller. The controller selects the subset of light sources accordingly. To determine the subset, the density of the fourier spectrum may be determined. If the fourier spectral density is small or no fourier data is present in certain regions, the corresponding light source is not used for measurement. Thus, the stacked imaging system enables content adaptive illumination.
According to another embodiment of the stacked imaging system, the subset of light sources to be operated is selected based on a previous calibration. Calibration includes generating a calibration image of the sample using all or most of the light sources. Further, a subset of light sources is selected based on their contribution to the calibration image.
According to another embodiment of the overlay imaging system, at the time of capturing the sub-image set, a subset of light sources to be operated is dynamically selected by determining the area and/or direction of the substantial signal contribution relative to a quality criterion and/or based on the signal strength, based on an evaluation of the signal content in fourier space of sub-images that have been taken in the sub-image set, by selecting light sources for subsequent images that are partially overlapping or adjacent to the area and/or direction of the substantial signal contribution that has been measured.
According to another embodiment of the stacked imaging system, the sub-images are captured by operating a plurality of light sources in parallel, by assigning substantial content in fourier space to the individual light sources operated, before selecting the light sources to be operated in subsequent frames and determining which of these light sources can be operated simultaneously, limited by a given maximum number and/or minimum distance criterion in the spatial or angular coordinate space of the light sources.
According to another embodiment of the stacked imaging system, the controller controls the operation of the plurality of light sources by adjusting illumination parameters of the light sources in dependence on their position within the arrangement of light sources. Controlling the illumination parameter may be based on a distance of the light source to the sample position and/or a tilt angle of the light source.
According to another embodiment of the stacked imaging system, adjusting the illumination parameter comprises adjusting at least one of:
a) the brightness of the light source is such that,
b) the duration of the operation of the light source,
c) the light-attenuating filter(s) may be,
d) a color filter is disposed on the substrate and has a color filter,
e) exposure time of the detector of a stacked imaging system, and
f) gain setting of a detector of a stacked imaging system.
The illumination parameters may be adjusted to a given signal-to-noise ratio of the detectors of the stacked imaging system. This means that the effective light flux, i.e. the amount of light, to the sample and/or from the sample to the detector is controlled. For example, the controller may control the drive current amplitude, the on-time of the drive current, the setting of the filter in the beam path; or exposure control of the detector may be performed by controlling the gain and/or exposure time. Preferably, the illumination parameters, such as at least one of the parameters a) to e), are controlled to achieve a given signal-to-noise ratio. Preferably, for each illumination angle, i.e. for each light source, a specific setting of the illumination parameter is determined.
According to another embodiment of the stacked imaging system, at least some of the control may be achieved by gain control, i.e. control of the sensitivity of the detector of the stacked imaging system.
According to another embodiment of the stacked imaging system, the controller adjusts the illumination parameter according to an azimuth angle of the light source.
The power density is proportional to the cosine of the angle of incidence (i.e., the azimuth angle of the light source). This means that more power is required for high angle light sources. Thus, the controller may adjust the illumination parameter depending on the azimuth angle of the light source, in particular compensating for such angular effects with the inverse cosine of the azimuth angle of the light source.
According to another embodiment of the overlay imaging system, the sub-images generated by the overlay imaging system are rescaled to a predetermined set of standard settings of the illumination parameters. The rescaling takes into account the individual scaling behavior of each illumination parameter, e.g. a linear scaling of the exposure time, a non-linear scaling of the LED driver current of the luminous flux, etc.
According to another embodiment of the stacked imaging system, the compensation is used in an adaptive manner according to a predetermined pattern of light sources. I.e. the compensation depends on the geometrical arrangement of the light sources. For example, the light sources may be arranged in planes with parallel optical axes or with individually tilted optical axes for at least one light source, wherein the optical axes are ideally directed towards the center of the field of view of the sample, i.e. the sample position. This arrangement reduces the amount of geometric correction of the geometric emission profile of the respective light source. Furthermore, the light sources may be arranged in a spherical geometry to minimize the effect caused by the distance of the light sources to the sample location. Furthermore, at least one of the light sources may be used and moved from one image capture to a different location for the next image capture in order to cover the entire aperture range of the desired image setting. This movement can be operated directly by physically moving the distal output ends of the individual light sources, such as LEDs or optical fibers; or indirectly by redirecting the light beam from the respective light source by, for example, a scanning unit such as a galvanometer scanner. Based on the respective geometrical settings of the light source with respect to the correct position, the controller determines and uses the adaptive settings for the correction.
According to another embodiment of the stacked imaging system, the controller controls the operation of the plurality of light sources to obtain different intensities at different diffraction angles (i.e. illumination apertures) to inherently provide contrast enhancement in the reconstruction process.
According to another embodiment of the method, controlling the operation of the plurality of light sources comprises the step of operating the plurality of light sources simultaneously. The number of light sources to be operated simultaneously is limited by a given maximum number and/or minimum distance criterion in the spatial or angular coordinate space of the light sources. For example, a minimum distance in the theta-phi space between an operating light source and its neighboring operating light source must be met.
According to another embodiment of the method, controlling operation of the plurality of light sources comprises selecting a subset of light sources of the plurality of light sources and operating only the light sources of the subset.
Drawings
The invention will be explained in more detail with reference to exemplary embodiments depicted in the drawings.
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments of the invention and together with the description serve to explain the principles of the invention.
Other embodiments of the present invention and many of the intended advantages of the present invention will be better understood by reference to the following detailed description. Like reference numerals designate corresponding similar parts. It should be understood that method steps are numbered for easier reference, but the numbering does not necessarily imply that the steps are performed in this order unless explicitly or implicitly described otherwise. In particular, the steps may also be performed in a different order than their numbered order indicates. Some steps may be performed simultaneously or in an overlapping manner.
FIG. 1 schematically shows a block diagram illustrating a stacked imaging system according to an embodiment of the invention;
FIG. 2 schematically illustrates a stacked imaging system according to an embodiment of the invention;
FIG. 3 shows the azimuth and pitch of the light source arrangement;
FIG. 4 shows a polar angle of a light source;
FIG. 5 shows the illumination numerical aperture of a planar array of light sources as a function of position along the x-axis;
FIG. 6 is a top view of an arrangement of light sources according to an embodiment of the invention;
fig. 7 schematically shows an arrangement of light sources according to fig. 6 in a side view;
FIG. 8 schematically illustrates a test object for simulation;
FIG. 9 illustrates a simulated low resolution image for grid-based conventional illumination;
FIG. 10 illustrates a reconstructed high resolution image for grid-based conventional illumination;
FIG. 11 illustrates a reconstructed high resolution image obtained by a stacked imaging system according to an embodiment of the invention;
FIG. 12 shows an exemplary Fourier spectrum of a test object;
FIG. 13 illustrates an exemplary subset of light sources for stack imaging;
FIG. 14 shows a reconstructed high resolution image obtained by a stacked imaging system according to an embodiment of the invention;
FIG. 15 shows a reconstructed high resolution image obtained by a stacked imaging system according to another embodiment of the invention;
FIG. 16 shows a reconstructed high resolution image obtained by a stacked imaging system according to yet another embodiment of the invention; and
FIG. 17 shows a flow diagram of a method of generating an image using a stacked imaging system, according to an embodiment of the invention.
Detailed Description
Fig. 1 shows a block diagram illustrating a stacked imaging system 1. The stacked imaging system 1 comprises a plurality of light sources 2, in particular Light Emitting Devices (LEDs), which emit light onto a sample arranged at a sample position of the stacked imaging system 1. The light sources 2 are arranged in a predetermined pattern. The overall shape of the arrangement may include a planar array or a spherical cap.
The light sources 2 may be arranged in a plurality of concentric rings around the central optical axis. All light sources 2 within the same concentric ring have the same azimuth angle measured between the central optical axis and the line from the light source 2 to the sample position. The azimuth angle of the light source 2 may exhibit a constant variation between adjacent concentric rings. In other words, the difference between the azimuth angles of the light sources 2 in different adjacent concentric rings is independent of the concentric ring under consideration.
Also, the change in polar angle may be uniform for every two light sources 2 selected within the same concentric ring. In other words, the difference between the polar angles of two adjacent light sources in the same concentric ring is independent of the selected light source 2. In particular, the light sources 2 in different concentric rings may have substantially the same polar angle variation, except for deviations due to the fact that only an integer number of light sources 2 are possible in each concentric ring.
The controller 3 of the stacked imaging system 1 controls the operation of the light source 2. The controller 3 may include at least one of a Central Processing Unit (CPU) or a Graphics Processing Unit (GPU) such as a microcontroller (μ C), an Integrated Circuit (IC), an Application Specific Integrated Circuit (ASIC), an Application Specific Standard Product (ASSP), a Digital Signal Processor (DSP), a Field Programmable Gate Array (FPGA), and the like. The controller 3 may also include or access memory. The memory may be a volatile or non-volatile data memory, such as a solid state disk, memory card, or the like. In a preferred embodiment, the controller may store a sequence of previously defined subsets of selected LEDs to be operated at a particular time and recall these subsets of the sequence during capture of sub-images of the entire set of sub-images.
The controller 3 activates at least one light source 2 at a time to generate a corresponding sub-image. The controller 3 may operate a subset of the plurality of light sources 2 simultaneously. In particular, for each sub-image, the controller 3 may select at least two light sources 2 to operate. The light sources 2 may be selected according to a predetermined pattern.
The controller 3 may also select a subset of the plurality of light sources 2. Only the selected light sources 2 are operated for generating the respective sub-images. One or more light sources 2 may be selected to generate each sub-image. The non-selected light sources 2 are not used to generate the sub-images. The controller 3 may select the light source 2 according to instructions stored in a memory.
The controller 3 may select the subset to be operated on in dependence on the characteristics of the sample to be observed. The overlay imaging system 1 may comprise a user interface for receiving instructions from a user. The user may select one or more options regarding the type of sample. For each type of sample, a respective subset of light sources 2 is selected by the controller 3.
The controller 3 may also select the light source 2 based on a previous calibration. During calibration, the controller 3 generates a low resolution sub-image. To generate the sub-images, all light sources 2 are used. In other words, there is no choice in the calibration phase. Then, the controller 3 may generate a high resolution image by combining the low resolution sub-images. The high resolution image is considered a calibration image. The controller 3 may then analyze the high resolution image using fourier analysis to determine the most contributing fourier components. For example, only those components whose average exceeds a particular threshold may be considered "most contributing" or relevant. A particular light source 2 of the plurality of light sources 2 corresponds to a respective fourier component. During operation, the controller 3 selects only the light source 2 corresponding to the fourier component identified during the calibration phase that contributes the most.
The controller 3 may be adapted to control the illumination parameters of the light source 2. The value of the illumination parameter may depend on the position of the light source 2 within the arrangement of light sources 2. In particular, the illumination parameters may be selected solely depending on the azimuth angle of the light source 2. In other words, the same illumination parameters are selected for light sources 2 having the same azimuth angle. At least some of the illumination parameters are different for light sources 2 having different azimuthal angles. The illumination parameters may include the brightness of the light source 2, the duration of operation of the light source 2, attenuation filters, color filters, gain values of the detectors of the stacked imaging system 1, and/or exposure time.
Fig. 2 illustrates an exemplary stacked imaging system 1. The stacked imaging system 1 comprises an array of light sources 2. The light source 2 emits (laser) light onto a sample plane 4 with a sample located at a sample position 5. The sample position is expanded. However, since the size of the sample position is small relative to the size of the distance between adjacent light sources 2, the sample position can be regarded as point-like in good approximation. The sample may comprise a solid structure or a fluid, such as a sample of blood.
The light emitted by the light source 2 interacts with the sample, e.g. via absorption, deflection or reflection. At least a portion of the light emitted by the light source 2 passes through the sample at the sample position 5 and enters the microscope objective 6. The imaging lens 7 focuses the light onto a real planar imaging detector 8. The detector 8 generates sub-images and stores the data, for example in a memory of the controller 3. The detector 8 may also provide the data to an external processing unit, such as an external computer for analyzing the data.
Although fig. 2 shows a uniform array of light sources 2, the array of light sources 2 may generally have a different arrangement, such as non-uniform spacing between the light sources 2. In particular, the arrangement may comprise a uniform variation of the azimuth angle and/or the polar angle. Furthermore, different arrangements may also include that different kinds of light sources may be mounted at different locations within the laminated luminaire. The different types of light sources may be selected from, but are not limited to, the following types: light Emitting Devices (LEDs) that emit in a single color or a single or multiple bandwidths, such as RGB LEDs, S-LEDs, lasers (especially semiconductor lasers), thermal emitters, optical fiber based light sources, etc. The light sources may differ in at least one of the following characteristics, for example: wavelength, spectral bandwidth, spatial emission characteristics, temporal emission characteristics such as continuous or pulsed operation, coherence parameters such as temporal and/or spatial coherence degree, brightness or etendue.
FIG. 3 shows the azimuth angle of the arrangement of the light source 2
And a spacing x. Azimuth angle
Is the angle measured between a central optical axis passing through the central light source 21 and extending to the sample position 5 and a line extending between the respective light source 22 and the sample position 5. The spacing x corresponds to the distance between two adjacent light sources 21, 22.
Fig. 4 shows the polar angle θ of the light source 2. The polar angle θ of a particular light source 22 corresponds to the angle between a line between that light source 22 and the central light source 21 and another line between a predetermined light source 23 and the light source 21. The predetermined light source 23 is arbitrarily selected as a reference. What is important is the change in the polar angle, i.e. the difference between the polar angles of two adjacent light sources 22, 23 within the same concentric circle, which is independent of the reference.
Fig. 5 shows the illumination numerical aperture a of a conventional uniform planar array of light sources 2 depending on the position B along the x-axis. Each source illumination provides a separate measurement in fourier space. This multiple separate measurements are used for reconstruction of high resolution high field of view images. For accurate reconstruction of the image, it is necessary to have at least 50% overlap between the fourier spectra measured by adjacent light emitting devices 2. In the center, the overlap between adjacent light emitting devices 2 may be about 55%. As can be seen from fig. 5, the illumination numerical aperture does not increase linearly. This causes an increase in the overlap between the fourier spectra of adjacent light sources 2 towards the periphery. To address this issue, a particular arrangement of the light sources 2 may be selected.
Fig. 6 schematically shows the arrangement of the light sources 2 in a top view.
Fig. 7 schematically shows a side view of the arrangement of the light source 2 according to fig. 6. The light sources 2 are arranged in concentric circles around a central optical axis. Azimuth angle
Is selected to be uniform and the variations of the polar angle theta between the light sources 2 in the same concentric circle are also selected to be substantially uniform. In other words, the stacked imaging system 1 includes a uniform theta-phi illuminator.
Preferably, the angular orientation of each concentric ring is statistically or randomly selected. In other words, additional symmetry is preferably avoided to prevent artifacts from occurring. Such artifacts may occur, for example, if there are large circles (or meridians) on the sphere that includes the light source 2 for each concentric ring. Preferably, the light sources 2 are not arranged along meridians.
Although fig. 6 and 7 illustrate a spherical uniform theta-phi illuminator, the light sources 2 may also be arranged in a plane, thereby providing a planar uniform theta-phi illuminator. In the case of a plane, additional symmetry is preferably also avoided. Thus, the LEDs of the different rings are not arranged on straight radial lines corresponding to the meridian of the spherical shell.
To determine this arrangement, first, the polar angle may be determined such that in the radial direction the continuous light source 2 has an overlap of about 50% in the fourier spectrum. These polar angles and the expected maximum numerical aperture are used to find the number of rings of the light source 2. Each ring will be stepped at a uniform polar angle to ensure that there is a uniform overlap in the radial direction.
For example, a desirable spacing for a numerical aperture of 0.1 may be about 5 mm. Most high power LEDs have a footprint of about 2 to 3 mm. If the height (i.e. the distance between the central light source 21 and the sample position 5) is chosen to be 70mm, there is an overlap of about 55% in the fourier spectrum. From the pitch x and the altitude, the azimuth angle can be calculated according to the following formula:
the position of the light source 2 along the axis is then determined based on the synthetic aperture requirements. For example, if the synthetic aperture needs to be 0.6, the illumination numerical aperture needs to be at least about 0.5, utilizing a numerical aperture of 0.1 for the lens by diffraction. The maximum azimuth angle can be calculated according to the following formula:
in the following, we will use
The radius of each circle can be calculated according to the following formula:
wherein n is 1, 2 …. The maximum radius depends on the desired effective numerical aperture. To obtain an illumination numerical aperture of 0.5, a central light source 2 surrounded by 7 rings with additional light sources 2 is required.
The radius of the circle is given by the following values (in mm):
4.8949,9.8379,14.8790,20.0722,25.4779,31.1660,37.2197。
the light sources 2 located at these radii will maintain a uniform overlap in the radial direction.
Next, it is described how the azimuth position of the light source 2 is found on each circle so that there is a uniform overlap in the azimuth direction.
First, for a change in the polar angle θ between adjacent light sources 2, the number of light sources 2 of the first circle is calculated using the following formula:
Δθ＝tan-1(pitch/radius) 45.6 °.
Thus, the first circle requires 360/45-8 light sources 2.
Likewise, the azimuth change is calculated for the respective circles to have the following values:
45.6086,26.9414,18.5746,13.9877,11.1031,9.1144,7.6512
the corresponding number of light sources 2 is calculated as
7.8933,13.3623,19.3813,25.7368,
32.4234,39.4981,47.0517,55。
In order not to violate the overlap criterion in the pupil by at least 50%, rounding to the nearest larger integer gives the number M of light sources 2 per circle n:
8,14,20,26,33,40,47,55.
angular step Δ θ of ring nnCalculated by the following formula:
Δθn＝360/Mn
wherein M isnRepresenting the corresponding number M of light sources in the ring n.
The position of each light source 2 can be found using the angular coordinates and radius using the following formula:
[x,y]＝[r·cos(m·Δθn),r·sin(m·Δθn)]。
in this formula, r denotes the radius of each light source 2, and M varies from 1 to the maximum number M of light sources 2 on the circle nn. According to a preferred embodiment for avoiding additional symmetry, for each ring n, a (possibly different) statistical angle Δ θ may be addedn0The statistical angle may be determined according to the following formula:
Δθn0＝Rn·Δθn
wherein the range [ 0; 1]Or preferably from the range [ 0; 1]Different random numbers of (2). Due to Delta thetanEven a random number will help to avoid additional symmetry. The position of the light source 2 will be found from the angular coordinate, the angular variation and the corresponding radius of the ring n by the following formula:
[x,y]＝[r·cos(m·Δθn+Δθn0),r·sin(m·Δθn+Δθn0)]
for the design of a spherical uniform theta-phi array, similar steps are used to find the polar and azimuthal angles. The only difference compared to a planar array is the determination of the coordinates of the light source using the following equation:
according to this formula, n varies from 1 to the maximum radius number. Delta thetanRepresenting the azimuth step in the number of rings n. m isnRepresenting the number of corresponding light sources 2 in the number of rings n, ranging from 1 to mn. For the preferred embodiment, the statistical angle Δ θ may be added accordinglyn0。
The light source in the arrangement described in the previous figures may comprise at least one of the following: semiconductor based light sources such as LEDs, SLEDs, semiconductor lasers (e.g. VCSELs), gas lasers, white light lasers (e.g. fiber crystal lasers), fluorescent or broadened based light sources (e.g. white light LEDs), thermal light sources (e.g. halogen lamps, arc lamps (e.g. Xe-Hg lamps)), optical fiber based light sources, wherein any of the aforementioned light sources can be coupled into an optical fiber and the output end of the optical fiber can be used as a light source. Furthermore, these light sources may be coupled with beam forming optics, such as collimators or focusing optics, to optimize the etendue of the light radiated onto the sample region 5 under investigation. The collimator optics may be selected from, but not limited to, the following: lenses, GRIN lenses, Diffractive Optical Elements (DOEs), refractive optical elements (e.g., fresnel lenses), computer generated holograms.
Fig. 8 schematically shows a test object for simulated ground truth. The simulation generates images of the test object for different stacked imaging systems, including conventional stacked imaging systems with planar uniform arrays of light sources 2.
Fig. 9 shows a simulated low resolution image of such a grid-based conventional illumination with a 0.1 numerical aperture objective. As can be observed, the quality is relatively poor and small structures are not resolved.
FIG. 10 illustrates a reconstructed high resolution image based on a mesh-based conventional illumination of a stitching of multiple low resolution images. A total of 289 low-resolution images are combined to obtain the high-resolution image shown in fig. 10.
FIG. 11 illustrates a reconstructed high resolution image obtained by a stacked imaging system 1 having a uniform theta-phi illuminator in accordance with the present invention. Due to the specific arrangement, only a small number of 244 images are required to reconstruct a high resolution image for the same numerical aperture as used by conventional imaging systems.
In the following, a stacked imaging system 1 is described, wherein the controller 3 operates the plurality of light sources 2 by selecting a subset of the light sources 2 of the plurality of light sources 2 and by operating only the selected light sources 2 of the subset.
Fig. 12 shows an exemplary fourier spectrum of the test object shown in fig. 8. It can be seen that the test object has significant fourier components only along the x-axis and y-axis. Thus, the number of light sources 2 to be operated can be limited without losing important information.
Fig. 13 shows an exemplary subset of light sources 2 for stack imaging. Only light sources 2 along the x-axis and along the y-axis are selected to generate the respective low resolution images.
Additionally or alternatively, a plurality of light sources 2 may be used simultaneously. For example, for each low resolution image, two light sources 2 may be selected and switched on to capture the respective low resolution image.
FIG. 14 illustrates a reconstructed high resolution image obtained by a stacked imaging system 1 having a uniform theta-phi illuminator in accordance with the present invention. The high resolution images are reconstructed from the low resolution images using multi-LED illumination with two light sources 2 for each low resolution image. High resolution images have been obtained by combining a total of 122 low resolution images in a stack reconstruction process.
FIG. 15 illustrates a reconstructed high resolution image obtained by a stacked imaging system 1 having a uniform theta-phi illuminator in accordance with the present invention. In order to generate low resolution images, adaptive selection of the content of the light source 2 has been employed. Therefore, only the light source 2 shown in fig. 13, i.e., only the light source 2 along the x-axis and the y-axis, is used to generate the low resolution image. The high resolution image is obtained by stitching a total of 31 low resolution images.
FIG. 16 shows a reconstructed high resolution image obtained by a stacked imaging system 1 having a uniform theta-phi illuminator in accordance with the present invention. In addition to multi-LED lighting using two light sources 2 simultaneously, content-adaptive selection of light sources 2 is also employed. The content adaptive selection may be based on a priori knowledge about the object. Therefore, only the light source 2 shown in fig. 13, i.e., only the light source 2 along the x-axis and the y-axis, is used to generate the low resolution image. Thus, the stacked imaging system 1 combines multiple LED illumination with content adaptive selection of the light sources 2. A high resolution image is obtained by reconstructing a total of 19 low resolution images.
In general, for a given object, the orientation of the pattern of fourier components in the pupil plane or in the fourier transform of the image, respectively, strongly depends on the orientation or three-dimensional pose of the object in the object space of the imaging system.
In fig. 16, the axes of the test pattern are aligned with the x-axis and y-axis of the sensor array of the imaging system. Furthermore, the quadratic grid of light sources is also aligned with respect to the x-axis and y-axis of the object, so the fourier pattern of the transformed image in the spatial frequency domain is aligned with the x-axis and y-axis of the detector, so the image frame and the light source to be selected are also selected to be aligned with the x-axis and y-axis of the grid of light sources 2 intersecting on the optical axis of the imaging system.
TABLE 1
Table 1 compares different stacked imaging systems 1. It can be seen that using each of the uniform theta-phi illuminator, the multi-LED illumination, and the content adaptive illumination may help reduce the number of images required for reconstruction. If all three measurements are used simultaneously, the number of images required for reconstruction is reduced from 289 to only 19. Thus, the time required to generate a low resolution image for reconstructing a high resolution image is significantly reduced.
In general, if the light sources are to be selected based on a priori knowledge of the image spatial frequency structure of the object, it is necessary to first determine the spatial orientation or pose of the object or first determine the geometrical main axes of a coordinate system representing the object in the object space of the imaging system. In a continuous step, a predetermined subset of the light sources 2 to be used in the imaging process is employed in terms of their angular orientation relative to the orientation or principal axis of the object when positioned in the object space of the imaging system.
Another alternative method for adaptive selection of the content of the light source 2 may be based on taking an image in a first step and on analysis of fourier components combined in the image in a second step. The light sources 2 may or may not be selected as part of a subset of the light sources 2 used in a subsequent replication of the imaging of the same object or the same kind of object in the imaging system based on the intensity of the contribution of the respective light source 2 to the signal when reconstructing the object. Furthermore, it is important that the measured orientation of the object is the same as the orientation of the same or reference object when the light source 2 is selected to be adapted to the spatial frequency content of the object. In case of any misalignment, the choice of light source 2 as described in the previous section for the case of a priori knowledge must be employed.
Content adaptive selection may also be based on iteratively examining higher order spatial frequency components in the object image. Preferably, the first image is taken with a low illumination aperture, such as an axis illumination based on the central light source 2 or a low aperture illumination from a light source with a small number n of rings. When determining the spatial frequency content of the image or of a subset of images with low aperture illumination, the light sources in the next larger number n of rings are preferably selected in those radial directions, where the substantial fourier components for the lower spatial frequencies have been selected. It may be determined based on a suitable measure whether the radial direction has a substantial fourier component, e.g. a threshold of the absolute intensity of the fourier component, or compared to adjacent regions of the respective region, or relative to an average of other fourier components having the same radial distance, or a desired background noise level for that radial distance. Since the method does not rely on reference information from a priori knowledge or the images taken in the first step, no angular correction is required when selecting the light sources 2 and applying them to successive images. On the other hand, an iterative inspection method may be beneficial for selecting the light sources 2 on the first object and continuously measuring comparable objects using the defined subset of light sources. Only when this subset of light sources 2 is applied to another object, the adopted angles of the light sources 2 selected for the respective orientation of the second object may be applied.
The content adaptive selection can be performed dynamically even when an image is taken. Initially, a first image is captured with a low illumination aperture angle, preferably for on-axis illumination. A fourier transform of the image is determined. In this fourier transform, the area or region or direction of the sequence of peaks in the radial axis is determined, wherein the substantial signal contribution is determined. Due to the overlap criterion of the fourier space selection light sources 2, those neighboring light sources 2 are selected whose respective illumination apertures overlap with the area, area or direction of the radial axis. The next image is taken for the light source or light sources, with the largest substantial signal contribution being in the overlapping area of the aperture space. When applying a plurality of light sources 2, the minimum distance criterion in the spatial or angular coordinate space of the light sources 2 may be followed. When the next image is taken, evaluation is performed similarly to the first image.
The next image is taken for the light source or light sources according to the area, region and/or direction of the determined radial axis of the two images, with the largest substantial signal contribution being in the overlapping area in aperture space. This procedure is performed for the other images until no substantial signal contribution is available anymore or if all remaining areas, areas or directions of the radial axis do not contribute any substantial contribution to the additional light source 2 not used so far. This situation may be considered a termination criterion. After the image subset is completed or the termination criteria is met, overlay evaluation is applied on the image subset to determine a high resolution image.
Fig. 17 shows a flow chart of a method of generating an image using the stacked imaging system 1. The method may be performed with any of the above-described stacked imaging systems 1.
The stacked imaging system 1 includes a plurality of light sources 2 arranged in a predetermined pattern. The light sources 2 may be arranged in a plurality of concentric rings around a central optical axis passing through the sample positions 5 of the stacked imaging system 1. The variation in the azimuth angle of the light sources in subsequent rings may be substantially uniform. Furthermore, the polar angle variation of adjacent light sources within the same concentric ring may be substantially uniform. The arrangement of the light sources 2 may be planar or spherical or any other geometry to support application specific optimization. For example, in a planar arrangement, the light sources may be tilted relative to the plane according to their respective distances from the optical axis. This arrangement then provides a non-uniform grid of individual light sources, each light source being tilted at its respective angle relative to the optical axis such that substantially all of the optical axes of all of the light sources intersect in a point or small region 5 in the sample plane. This arrangement helps to minimize the effect of spatially non-uniform emission characteristics of the light source 2 as is typically seen on more directional emission profiles of, for example, LEDs, optical fibers, fiber bundles, lasers with slow and fast axes, or VCSELs.
In a first method step S1, the controller 3 sends a control signal to the light source 2 in order to operate the light source 2 to emit (laser) light onto the sample at the sample position 5 of the stack imaging system 1.
The controller 3 may control the operation of the plurality of light sources 2 by simultaneously operating the plurality of light sources 2. In other words, each low-resolution image is generated by using the plurality of light sources 2. Each low resolution image may be obtained, for example, by using two light sources 2.
The controller 3 may also control the operation of the plurality of light sources 2 by selecting a subset of the light sources 2 of the plurality of light sources 2 and by operating only the light sources 2 of the subset. For example, the controller 3 may select the light sources 2 only along the x-axis and the y-axis, as shown in fig. 13. The selection of the light source may be based on the characteristics of the sample to be observed or measured. The selection may also be based on user input. For example, the user may select between several types of samples, and the controller 3 selects the light source 2 corresponding to the selected type of sample. Which light sources 2 can be stored in a look-up table in the memory of the stacked imaging system 1 are selected for the respective type of sample. The controller 3 uses a look-up table to select the light source 2. A sample type specific look-up table may be provided as a preset to the user.
The subset of light sources 2 may also be selected based on a previous calibration. During calibration, one or more light sources 2 are used to generate a low resolution image. All light sources 2 or at least a majority of the light sources 2 are used to generate the respective low resolution images. Finally, the low resolution images are combined to generate a calibration image. The calibration image is analyzed, for example using fourier analysis, in order to determine the most significant contribution of the light source 2. For example, only the light sources 2 corresponding to fourier components that contribute to the fourier spectrum of the calibration image and exceed a predetermined threshold are selected. This selection of "useful" light sources can also be performed dynamically in real time during the image capture process of the low resolution image to minimize and optimize image capture and skip illumination in areas adjacent to the LEDs where no significant contribution in the fourier component is measured or where the corresponding intensity is very low relative to the background level.
The controller 3 may also control the operation of the plurality of light sources 2 by adjusting the lighting parameters of the light sources 2 in dependence on the position of the light sources 2 within the arrangement of light sources 2. In particular, the controller 3 may adjust the brightness of the light source 2, the duration of operation of the light source 2, attenuation filters, color filters, gain of the detectors of the stacked imaging system 1, and/or exposure time.
The illumination parameters may be adjusted to reduce or eliminate geometric effects due to the arrangement of the light sources 2. For example, the illumination parameter may be adjusted depending on the azimuth angle of the light source 2, e.g. depending on the cosine of the azimuth angle of the light source 2.
In a further method step S2, a plurality of low-resolution images generated by one or more light sources 2 are combined in order to generate a high-resolution image.
In a further method step, the high-resolution image may be further analyzed, for example, in order to detect specific components or structures in the high-resolution image. The analysis may employ standard image analysis tools.
It is to be understood that all advantageous alternatives, modifications and variations described herein as well as the aforementioned variations with respect to the embodiments of the stacked imaging system according to the first aspect may equally be applied to the embodiments of the method according to the second aspect and vice versa.
In the foregoing detailed description, various features are grouped together in one or more examples for the purpose of streamlining the disclosure. It is to be understood that the above description is intended to be illustrative, and not restrictive. It is intended to cover alternatives, modifications, and equivalents. Many other embodiments will be apparent to those of skill in the art upon reading the above description.
List of reference numerals
1 laminated imaging system
2 light source
3 controller
4 sample plane
5 sample position
6 microscope objective
7 imaging lens
8 imaging detector
21-23 light source
Numerical aperture of A illumination
Position B
x distance
Theta polar angle
S1 first method step
S2 second method step.
Claims (17)
1. A laminated imaging system (1) comprising
A plurality of light sources (2) adapted to emit light onto a sample location (5), wherein the light sources (2) are arranged in a predetermined pattern; and
a controller (3) adapted to control the operation of the plurality of light sources (2);
wherein at least one of a) the predetermined pattern of light sources (2) and b) the control operation of the plurality of light sources (2) is adapted to compensate for geometrical effects due to the arrangement of the light sources (2) with respect to the sample position (5).
2. The stacked imaging system (1) as defined in claim 1, wherein the predetermined pattern of the plurality of light sources (2) comprises the light sources (2) arranged in a plurality of concentric rings around a central optical axis passing through the sample position (5);
wherein the variation of the azimuth angle is substantially uniform for the light source (2) in the subsequent ring, wherein the azimuth angle of the light source (2) is measured between the central optical axis and a line from the light source (2) to the sample position (5); and
wherein the change in polar angle is substantially uniform for adjacent light sources (2) within the same concentric ring.
3. The stack imaging system (1) according to claim 1 or 2, wherein the arrangement of the light sources (2) is planar.
4. The stacked imaging system (1) according to claim 1 or 2, wherein said arrangement of said light sources (2) is spherical.
5. The stacked imaging system (1) according to any one of the preceding claims, wherein the controller (3) is adapted to control the operation of the plurality of light sources (2) comprising the step of simultaneously operating the plurality of light sources (2), wherein the number of light sources (2) to be simultaneously operated is limited by a given maximum number and/or minimum distance criterion in the spatial or angular coordinate space of the light sources (2).
6. The stack imaging system (1) according to any one of the preceding claims, wherein the controller (3) is adapted to control the operation of the plurality of light sources (2) comprising the step of selecting a subset of light sources (2) of the plurality of light sources (2) and operating only the light sources (2) of the subset.
7. The overlay imaging system (1) according to claim 6, wherein the subset of light sources (2) to be operated is selected based on characteristics of a sample to be observed.
8. The overlay imaging system (1) according to claim 6 or 7, wherein the subset of light sources (2) to be operated is selected based on a user input.
9. The overlay imaging system (1) according to claim 6, wherein the subset of light sources (2) to be operated is selected based on a previous calibration, wherein the calibration comprises the steps of:
generating a calibration image of the sample using all light sources (2); and
selecting the subset of light sources (2) based on a contribution of the light sources (2) to the calibration image.
10. The overlay imaging system (1) according to claim 6, wherein the subset of light sources (2) to be operated is dynamically selected at the time of capturing the set of sub-images by determining the area and/or direction of the substantial signal contribution relative to a quality criterion and/or based on signal strength based on an evaluation of the signal content in Fourier space of sub-images that have been taken in the set of sub-images, by selecting light sources (2) for subsequent images that are partially overlapping or adjacent to the area and/or direction of the substantial signal contribution that has been measured.
11. The overlay imaging system (1) according to claim 10, wherein the sub-images are captured by operating a plurality of light sources (2) in parallel, by assigning a substantial content in fourier space to the respective light sources operated, before selecting light sources (2) to be operated in subsequent frames and determining which of these light sources (2) can be operated while being limited by a given maximum number and/or minimum distance criterion in the spatial or angular coordinate space of the light sources (2).
12. The stack imaging system (1) according to any one of the preceding claims, wherein the controller (3) is adapted to control the operation of the plurality of light sources (2) comprising the step of adjusting an illumination parameter of the light source (2) depending on the position of the light source (2) within the arrangement of light sources (2).
13. The stack imaging system (1) according to claim 12, wherein adjusting the illumination parameter comprises adjusting at least one of: brightness of the light source (2), duration of operation of the light source (2), attenuation filters, color filters, gain of detectors of the stacked imaging system (1), and exposure time.
14. The stack imaging system (1) according to any one of claims 12 or 13, wherein the controller (3) is adapted to adjust the illumination parameter depending on an azimuth angle of the light source (2).
15. A method for generating an image using a stacked imaging system (1), comprising a plurality of light sources (2) arranged in a predetermined pattern, the method comprising:
emitting light by the plurality of light sources (2) onto a sample location (5) comprising a sample; and
controlling the operation of the plurality of light sources (2) by a controller (3);
wherein at least one of a) the predetermined pattern of light sources (2) and b) the control operation of the plurality of light sources (2) is adapted to compensate for geometrical effects due to the arrangement of the light sources (2) with respect to the sample position (5).
16. The method according to claim 15, wherein controlling the operation of the plurality of light sources (2) comprises the step of operating the plurality of light sources (2) simultaneously, wherein the number of light sources (2) to be operated simultaneously is limited by a given maximum number and/or minimum distance criterion in a spatial or angular coordinate space of the light sources (2).
17. The method according to any one of claims 15 or 16, wherein controlling the operation of the plurality of light sources (2) comprises the step of selecting a subset of light sources (2) of the plurality of light sources (2) and operating only the light sources (2) of the subset.
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